Endothelial cells play a unique role in modulating vascular tone by releasing endothelium-derived relaxing factor (EDRF). Defects in EDRF may predispose the diabetic to a higher risk of hypertension, atherosclerosis, coronary artery vasospasm and sudden death. This proposal will examine the hypothesis that diabetes selectively uncouples relaxation to receptor-operated endothelium-dependent vasodilators (RO-EDV), but not receptor-independent endothelium vasodilators (RI-EDV). The specific role oxygen-derived free radicals (ODFR)in this defect will be investigated. Blood vessels (aorta, femoral and mesenteric artery) from streptozotocin-diabetic and genetically-diabetic BB rats will be used and compared to glucose- infused rats. Selectivity and specificity for impaired response to RO- EDV vs. RI-EDV and endothelium-independent vasodilators will be examined (Specific Aim #1). In vivo intervention (Specific Aim #2) using surgery (pancreatic transplantation) or therapeutic intervention (insulin, antioxidant, free radical scavengers, iron chelator) will be evaluated to test for prevention or reversal of endothelial dysfunction. The mechanism of impaired EDRF will be examined (Specific Aim #3). The bioassay technique will be utilized in which endothelial- perfusion of control or experimental donor segments are challenged with agonists which release EDRF and acts upon detector rings (without endothelium). This will answer: Does the diabetic endothelium release less EDRF?; Is the diabetic vascular smooth muscle less responsive to authentic EDRF and/or nitric oxide?; Do ODFR produced by the diabetic endothelium or diabetic vascular smooth muscle impair or inactivate EDRF activity? The influence of insulin on intracellular glucopenia or EDRF release or action will also be tested. Additional protocols will test for a potential endothelium-derived constricting factor or differential changes in cGMP (using radioimmunoassay). Superoxide dismutase, catalase and glutathione peroxidase activities will be examined in blood vessels to assess radical scavenging capacity. Production of ODFR by blood vessels (with or without endothelium) will be examined by spectrophotometric techniques and by state-of-the art electron spin resonance spectroscopy using loop-gap resonators with spin trapping techniques. Intracellular calcium signal transduction in response to RO-EDV and RI-EDV will be evaluated using fluorescence spectroscopy (Fura-2) in cultured bovine aortic endothelial cells subjects to conditions mimicking the diabetic environment (Specific Aim #4). Endothelial cells will be incubated with either glucose, fatty acids, ketone bodies or diabetic sera (including very low density lipoproteins) to examine selective uncoupling of intracellular calcium signal transduction in response to RO-EDV. The role of ODFR in this uncoupling will also be evaluated. Data obtained from this study will help define a mechanism for defective EDRF and understanding the role EDRF may play in the etiology of hypertension and other vascular complications associated with diabetes. It will also provide experimental evidence for the efficacy of pancreatic transplantation or therapeutic intervention to prevent or reverse endothelial dysfunction.